The purified RNA is analysed for quality (by capillary electrophoresis) and quantity (for example, by using a NanoDrop or NanoPhotometer spectrometer). If the material is of acceptable quality and sufficient quantity is present (e.g., >1μg, although the required amount varies by microarray platform), the experiment can proceed.
The labelled product is generated via reverse transcription and sometimes with an optional PCR amplification. The RNA is reverse transcribed with either polyT primers which amplify only mRNA or random primers which amplify all RNA which is mostly rRNA, miRNA microarray ligate an oligonucleotide to the purified small RNA (isolated with a fractionator) and then RT and amplified. The label is added either in the RT step or in an additional step after amplification if present. The sense that is labelled depends on the microarray, which means that if the label is added with the RT mix, the cDNA is on the template strand while the probe is on the sense strand (unless they are negative controls). The label is typically fluorescent; only one machine uses radiolabels. The labelling can be direct (not used) or indirect which requires a coupling stage. The coupling stage can occur before hybridization (two-channel arrays) using aminoallyl-UTP and NHS amino-reactive dyes (like cyanine dyes) or after (single-channel arrays) using biotin and labelled streptavin. The modified nucleotides (typically a 1 aaUTP: 4 TTP mix) are added enzymatically at a lower rate compared to normal nucleotides, typically resulting in 1 every 60 bases. The aaDNA is then purified with a column (using solution containing phosphate buffer as Tris contains amine groups). The aminoallyl group is an amine group on a long linker attached to the nucleobase, which reacts with a reactive dye. A dye flip is a type of replicate done to remove any dye effects in two-channel dyes, in one slide one same is labeled with Cy3 the other with Cy5, this is reversed in a different slide. In this example, in the presence of aminoallyl-UTP added in the RT mix.
This mix is denatured and added to a pin hole in a microarray, which can be a gene chip (holes in the back) or a glass microarray which is bound by a cover, called a mixer, containing two pinholes and sealed with the slide at the perimeter.
The holes are sealed and the microarray hybridized, either in a hyb oven, where the microarray is mixed by rotation, or in a mixer, where the microarray is mixed by alternating pressure at the pinholes.
After an overnight hybridization, all nonspecific binding is washed off (SDS and SSC).
The microarray is dried and scanned in a special machine where a laser excites the dye and a detector measures its emission.
The image is gridded with a template and the intensities of the features (several pixels make a feature) are quantified.
The raw data is normalized, the simplest way is to subtract the background intensity and then divide the intensities making either the total intensity of the features on each channel equal or the intensities of a reference gene and then the t-value for all the intensities is calculated. More sophisticated methods include z-ratio, loess and lowess regression and RMA (robust multichip analysis) for Affymetrix chips (single-channel, silicon chip, in situ synthesised short oligonucleotides).